Saponins from Zygophyllum gaetulum - Journal of Natural Products

Three new bisdesmosidic triterpene saponins, zygophylosides I (1), L (2) and M (3), and three known ... Journal of Natural Products 2011 74 (10), 2116...
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J. Nat. Prod. 1998, 61, 130-134

Saponins from Zygophyllum gaetulum Omar Safir and Souad Fkih-Tetouani Laboratoire de Chimie des Plantes et de Synthe` se Organique et Bioorganique, Faculte´ des Sciences, Rabat, Morocco

Nunziatina De Tommasi Dipartimento di Scienze Farmaceutiche, Universita` di Salerno, Salerno, Italy

Rita Aquino* Dipartimento di Chimica delle Sostanze Naturali, Universita` “Federico II”, via D. Montesano 49, 80131 Napoli, Italy Received May 12, 1997X

Three new bisdesmosidic triterpene saponins, zygophylosides I (1), L (2) and M (3), and three known quinovic acid glycosides were isolated from Zygophyllum gaetulum. The new compounds were determined to be 3β-O-R-L-rhamnopyranosyl-(1f2)-R-L-arabinopyranosyl-(1f2)-β-Dglucopyranosylurs-20(21)-en-28-oic acid 28-O-[β-D-2-O-sulfonylglucopyranosyl] ester (1), 3β-O[R-L-rhamnopyranosyl-(1f2)-R-L-arabinopyranosyl-(1f2)-β-D-glucopyranosyl]urs-20(21)-en-28oic acid 28-O-[β-D-glucopyranosyl] ester (2), and 3β-O-β-D-quinovopyranosyl-27-nor-olean-12en-28-oic acid 28-O-β-D-glucopyranosyl ester (3) by use of 1D and 2D NMR techniques. In the course of our research program on African medicinal plants, we investigated Zygophyllum gaetulum Emb. & Maire (Zygophyllaceae), vernacular name “aggaya”, a species endemic in the Moroccan Sahara. The aqueous extracts of the aerial parts are used in the indigenous system of medicine as an antispasmodic, antieczema, and antidiabetic drug, and as a remedy for stomach and liver pain.1 No previous investigation is reported on this species. In this paper we describe the isolation and elucidation of three new bisdesmosidic saponins, compounds 1 (zygophyloside I), 2 (zygophyloside L), and 3 (zygophyloside M). The related known glycosides, 3β-O-β-D-glucopyranosylquinovic acid 28-Oβ-D-glucopyranosyl ester,2 zygophyloside G,3 and zygophyloside E4 were also isolated. The presence of triterpenes of the ursane series and of -SO3H moiety in the metabolites from Zygophyllum species has already been reported3-6 and seems to be of chemotaxonomic significance. Six saponins were isolated and purified by Sephadex LH-20 column chromatography and reversed-phase HPLC from the MeOH extract of the aerial part of Z. gaetulum. Zygophylosides I (1) (C53H86O24S) and M (2) (C53H86O21) gave quasi-molecular ion peaks at m/z 1137 [M - H]- and m/z 1057 [M - H]-, respectively, and prominent peaks due to the loss of a hexose for 2 and a hexose plus -SO3 for 1 in their negative FABMS. The NMR spectral data of saponins 1 and 2 (Tables1-3) revealed the feature of a triterpenic acid with a double bond and four sugar units, one of which was linked to a carboxyl group (δH 5.42, δC 92.83 in 1) via ester linkage.2 The remaining sugars were attached to the oxygen at C-3. * To whom correspondence should be addressed. Phone: 39 81 7486540. FAX: 39 81 7486510. X Abstract published in Advance ACS Abstracts, December 15, 1997.

S0163-3864(97)00245-0 CCC: $15.00

The 1H-NMR spectra of 1 and 2 displayed signals for six tertiary Me groups (δ 0.85, 0.90, 1.00, 1.01, 1.08, and 1.63) and one secondary Me group (δ 1.04, d, J ) 6.5 Hz) in the aglycon moiety, indicating a pentacyclic triterpene skeleton of the ursane or taraxastane series;7-10 one of the singlet Me signals appeared at comparatively lowfield (δ 1.63), suggesting that it could be attached with an olefinic carbon. In addition it was possible to observe an olefinic proton signal at δ 5.27 (1H, dd, J ) 6.0 and 1.5 Hz) and a signal typical of H-3ax (δ 3.20, dd, J ) 11.1, 4.5 Hz) due to the presence of β-OH group at C-3 position (Table 1). Full assignments of the proton and carbon signals of the aglycon part of 1 were secured by 1H-1H DQFCOSY11 and HSQC12 spectra. A combination of COSY and HSQC experiments showed the following connectivities C-3-C-2-C-1 for ring A, C-5-C-6-C-7 for ring B, C-16-C-15 for ring D, and C-13-C-12-C-11-C-9 starting from the well-resolved signals at δ 3.20 (H-3), 0.75 (H-5), 2.31 (H-16eq), and 2.42 (H-13). The proton and carbon signals due to the A, B, C, and D rings indicated a 3-β-hydroxy-12,13-dihydro-ursane skeleton carrying a -COOR group at C-28 and glycosylated at C-3.7-10 The position of the double bond on ring E at C-20(21) was established by the following evidence: there was only one proton (δH 5.27) connected to the olefinic carbons (δC 142.00 and 118.26), supporting the structure CHdC. The CH3 signal at δH 1.63 (s, Me-30) indicated a vinylic Me, the other (Me-29) at δ 1.04 (d, J ) 6.5 Hz) appeared shifted downfield by ca. +0.08 ppm with respect to the doublet Me signals typical of ursolic acid skeleton,2 due to the presence of the double bond. The COSY spectrum indicated that the olefinic proton was connected to a methylene group at 1.82 (dd, J ) 12.0, 1.5 Hz) and 2.36 (dd, J ) 12.0, 6.0 Hz), which correlated by HSQC to a CH2 signal at δC 37.89, ascribable to position C-22. The above-mentioned properties left only position C-20(21) for the vinylic double

© 1998 American Chemical Society and American Society of Pharmacognosy Published on Web 01/23/1998

Notes

Journal of Natural Products, 1998, Vol. 61, No. 1

Table 1. 13C-NMR and1H-NMR Assignments and HSQC, and HMBC Experiments in CD3OD carbons

δC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

40.09 27.06 91.47 40.34 57.16 19.16 33.08 42.06 51.99 38.08 22.60 30.29 40.21 42.80 28.61 33.02 50.57 50.26 38.31 l42.0 118.26 37.89 16.73 28.31 16.40 16.91 15.24 176.33 23.84 21.98

Long-range Correlations of Aglycon of Compound 1 by 1H-1H COSY,

δH (JHH in Hz)

DEPT CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH2 CH C CH2 CH2 C CH CH C CH CH2 CH3 CH3 CH3 CH3 CH3 C CH3 CH3

13C-1H

δC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

39.84 28.00 90.54 40.35 56.83 19.89 32.74 43.0 51.71 38.01 23.57 30.68 40.0 42.90 28.07 33.37 50.64 50.11 38.13 141.98 117.94 38.04

CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH2 CH C CH2 CH2 C CH CH C CH CH2

23 24 25 26 27 28 29 30

16.38 28.07 16.28 16.55 14.95 176.55 23.58 21.68

CH3 CH3 CH3 CH3 CH3 C CH3 CH3

cross peaks (δC) in HMBC spectrum

1.17 m, 1.88 m 1.08 m, 1.80 m 3.20 dd (11.1, 4.5)

16.73 (C-23), 28.31 (C-24), 4.49 (Glc-1′)

0.75 m 1.35 m, 1.55 m 1.36 m, 1.53 m

16.73 (C-23), 19.16 (C-6), 33.08 (C-7)

1.37 m 1.73 m, 1.95 m 1.22 m, 1.32 m 2.42 dt (12.0, 3.4) 1.05 m 1.11 m 1.36 ddd (13.0, 12.0, 5.0) 2.31 ddd (12.0, 5.0, 3.1) 1.23 br s 2.16 dd (11.5, 6.5) 5.27 dd (6.0, 1.5) 1.82 dd (12.0,1.5) 2.36 dd (12.0, 6.0) 0.85 s 1.08 s 1.01 s 0.90 s 1.00 s

50.26 (C-18), 118.26 (C-21), 142.0 (C-20) 27.06 (C-2), 40.34(C-4), 57.16 (C-5) 16.73 (C-23), 40.34 (C-4), 57.16 (C-5) 40.34 (C-4), 51.99 (C-9) 38.08 (C-10), 40.21 (C-13), 57.16 (C-5) 28.61 (C-15), 33.08 (C-16), 42.06 (C-8), 42.80 (C-14)

1.04 d (6.5) 1.63 s

50.26 (C-18), 142.0 (C-20) 38.31 (C-19), 118.26 (C-21), 142.0 (C-20)

Table 2. 13C-NMR and1-H NMR Assignments of Aglycon of Compound 2 by 1H-1H COSY and HSQC Experiments in CD3OD carbons

131

δH (JHH in Hz)

DEPT

1.02 m, 1.72 m 1.05 m, 1.78 m 3.20 dd (11.2, 4.5) 0.75 m 1.10 m, 1.55 m 1.32 m, 1.50 m 1.40 m 1.35 m, 1.92 m 1.27 m, 1.33 m 2.41 dt (12.2, 3.4) 1.05 m, 1.27 m 1.45 br 2.10 ddd (12.2, 5.2, 3.2) 1.26 br 2.15 dd (11.5, 6.5) 5.27 dd (6.0, 1.5) 1.87 dd (12.2, 1.5), 2.32 dd (12.2, 6.2) 0.85 s 1.08 s 1.00 s 0.90 s 1.03 s 1.04 d (6.5) 1.63 s

bond. The value of the coupling constant between H-18 and H-19 (J ) 11.5 Hz) indicated that both protons were in an axial position; therefore, the Me at C-19 should be in β-position and rings D/E should have a cis connection.

This interpretation was unambiguously confirmed by the HMBC13 (8 Hz) spectrum of 1, which showed significant cross peaks, due to 2JC-H and 3JC-H correlations, between H-22ax (δ 2.36) and C-21, C-20 and C-18, between the protons of Me-29 (δ 1.04) and C-18 and C-20, and between the protons of Me-30 (δ 1.63) and C-19, C-20, and C-21. Similar correlations between H-3 (δ 3.20) and Me-23, Me-24 and C-1 of a glucose unit led us to confirm the proposed structure. The stereochemistry at C-17,18 (cis linkage of D/E rings) was authenticated by the ROESY14 spectrum, which showed key correlation peaks between H-19 (δ 2.16) and the signals at δ 1.01 (Me-27) and 1.63 (Me30), between H-13 (δ 2.42) and Me-25 (δ 1.00), H-18 (δ 1.23) and H-16eq (δ 2.31), as well as between H-21 (δ 5.27), H-22ax (δ 2.36), and Me-30 (δ 1.63) signals. In the triterpenoids of the taraxastane series where the D/E ring junction is trans, no such interactions could be observed.9,10 Other diagnostic ROEs were recorded between H-5R and H-3R and between H-1′ of the glucose and both H-2′ of the glucose and H-3 of the aglycon. Therefore, the structure of the aglycon of saponin 1 is 3β-urs-20(21)-en-28-oic acid. To the best of our knowledge this is the first report of a naturally occurring pentacyclic triterpene of the ursane series with a double bond at C-20(21) and a -COOH at C-28; however, ∆12 or ∆18 or ∆12,18 or ∆18,20 ursanes have been previously described.15 Its isolation from Z. gaetulum may be taxonomically significant. Four anomeric signals at δH 4.49, 5.00, 5.05, and 5.52 were easily identified in the 1H spectrum of 1. They correlated to carbons at δC 105.65, 101.41, 102.15, and 92.83, respectively, in the HSQC spectrum. The chemical shift of the fourth signal showed that this sugar moiety was attached to C-28 of the aglycon by an ester

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Table 3.

13C-NMR

Notes

and1H-NMR Dataa of Sugar Moiety of Compounds 1 and 2 in CD3OD 1

positionc

δC

Glc-1′ Glc-2′ Glc-3′ Gl-4′ Glc-5′ Glc-6′

105.65 79.46 76.56 73.38 76.64 61.88

Ara-1′′ Ara-2′′ Ara-3′′ Ara-4′′ Ara-5′′

101.41 75.54 72.36 67.43 63.37

Rha-1′′′ Rha-2′′′ Rha-3′′′ Rha-4′′′ Rha-5′′′ Rha-6′′′ Glc-1′′′′ Glc-2′′′′ Glc-3′′′′ Glc-4′′′′ Glc-5′′′′ Glc-6′′′′

102.15 72.09 71.82 73.65 69.86 17.96 92.83 79.52 77.30 70.74 78.04 61.88

2 δH (JHH in

Hz)b

4.49 d (7.8) 3.42 dd (9.0, 7.8) 3.63 t (9.0) 3.52 t (9.0) 3.64 ddd (9.0, 4.5, 3.0) 3.70 dd (12.0, 4.5) 3.85 dd (12.0, 3.0) 5.00 d (5.2) 3.92 dd (5.2, 8.5) 3.82 dd (8.5, 3.0) 3.80 m 3.45 dd (12.0, 3.0) 3.95 dd (12.0, 2.0) 5.05 d (1.5) 3.88 dd (1.5, 2.0) 3.71 dd (2.0, 8.5) 3.45 t (8.5) 3.87 dd (8.5, 6.6) 1.30 d (6.6) 5.52 d (7.6) 4.20 dd (9.0, 7.5) 3.76 t (9.0) 3.53 t (9.0) 3.40 m 3.73 dd (12.2, 5.0) 3.84 dd (12.2, 3.5)

δC

δH (JHH in Hz)b

105.47 79.57 76.47 73.52 77.71 62.13

4.48 d (7.5) 3.43 dd (9.0, 7.5) 3.60 t (9.0) 3.51 t (9.0) 3.60 ddd (9.0, 4.5, 3.5) 3.72 dd (12.0, 4.5) 3.84 dd (12.0, 3.5) 5.03 d (5.2) 3.92 dd (5.2, 8.5) 3.83 dd (8.5, 3.0) 3.82 m 3.45 dd (12.0, 2.5) 3.97 dd (12.0, 2.00) 5.05 d (1.5) 3.88 dd (1.5, 2.5) 3.71 dd (2.5, 8.6) 3.41 t (8.6) 3.88 dd (8.6, 6.5) 1.30 d (6.5) 5.47 d (7.5) 3.30 dd (9.5, 7.5) 3.43 t (9.5) 3.57 t (9.5) 3.37 m 3.70 dd (12.2, 4.5) 3.85 dd (12.2, 3.5)

101.17 75.69 72.32 67.41 63.30 101.97 72.12 71.91 73.67 69.94 18.00 94.89 73.98 78.07 70.98 78.33 62.13

a Assignments confirmed by 1D TOCSY and 2D COSY, HSQC, HMBC experiments. b 1H-1H coupling constants in the sugar spinspin were measured from TOCSY and COSY spectra in Hz. c Glc ) β-D-glucopyranosyl, Ara ) R-L-arabinopyranosyl, Rha ) R-L-rhamnopyranosyl.

bond. The sugars were determined to be a trisaccharide chain formed by glucose, arabinose, and rhamnose linked to C-3 of the aglycon, and a 2-O-sulfonyl-glucose at C-28 by use of monodimensional TOCSY,16 2D DQFCOSY, and HSQC experiments. Even at highfield (600 MHz) the 1D sugar spectral region of 1 was complex as most of the shifts were overlapped and found between δ 4.20 and 3.30. The isolated anomeric proton signals resonating at an uncrowded region of the spectrum (between δ 4.49 and 5.52) have been the starting point for the 1D TOCSY experiments. Because of the selectivity of the multistep coherence transfer, the 1D TOCSY subspectra of the single monosaccharide unit could be extracted from the crowded overlapping region. Each subspectrum could be attributed to one set of coupled protons such as H-C (1) to H-C (4) (for arabinose) or H-C (6) (for glucose and rhamnose) of an individual monosaccharide. The 1D TOCSY subspectra of the four monosaccharide units could be interpreted and, at the same time, the type of sugar, its configuration, and conformation assigned by the 2D COSY spectrum, as summarized in Table 3. The HSQC spectrum led to establish the position of the interglycosydic linkages by comparison of observed resonances with those of the corresponding methylpyranosides and accounting for the known effects of glycosydation.17 Once the proton and carbon spectra had been completely assigned, an unambiguous determination of the sequence and linkage sites was obtained from the longrange C-H correlations (HMBC spectrum) and from the ROEs in the 2D ROESY spectrum. The esterified sugar substituent at C-28 was identified from the following evidence: a 1D TOCSY subspectrum obtained by irradiating at δ 5.52 showed a set of coupled protons at δ 4.20, 3.76, 3.53, 3.40 (all CH), and 3.73 and 3.84 (CH2) assigned as H-1′′′′ to H2-6′′′′ of a

2-O-esterified glucopyranose by the COSY spectrum. Analysis of the correlated 13C-NMR signals in the HSQC spectrum led to the identification of a 2-substituted glucopyranose linked at C-28 by an ester bond. The characteristic downfield shift3,4 of H-2′′′′ (δ 4.20) and C-2′′′′ (δ 79.52) indicated the location of the -SO3H group to this position. The cross peak of the 3J longrange coupling between H-1′′′′ of the 2-O-sulfonylglucopyranose and C-28 of the aglycon gave definitive evidence for the position of this sugar moiety. The presence of the -SO3H group was confirmed by solvolysis of 1, which yielded compound 2 (see Experimental Section). The other three monosaccharide units were connected together, and one showed correlation (HMBC) with C-3 of the aglycon. The absence of any glycosylation shift for the carbon resonances of the rhamnopyranose suggested this sugar to be the terminal unit, while glycosylation shifts were observed for C-2 (ca. 6 ppm) of the arabinopyranose and glucopyranose units. The 1H-NMR as well as 13C-NMR data of key carbons (C-2, C-3, and C-5), indicated a β-configuration at the anomeric positions of the two D-glucopyranosyl units (JH1-H2 ) 7.5 Hz) and an R-configuration for the L-rhamnopyranose (JH1-H2 ) 1.5 Hz). L-Arabinose in the pyranose form was evident from 13C-NMR data; however, the value of its JH1-H2 coupling constant (5.2 Hz), midway between that reported for methyl-R-arabinopyranoside (4 Hz) and methyl-β-arabinopyranoside (8 Hz), was not diagnostic owing to the high conformational mobility of arabinopyranosides (4C1T1C4). As we also reported in previous work,18,19 evidence of R-Larabinopyranoside was obtained from ROESY spectrum, which showed NOEs from C-1′′ to C-2′′, C-3′′, and C-5′′ indicative of R-L-arabinopyranoside in rapid conformational exchange from 4C1T1C4 conformation.

Notes

Figure 1.

Key correlation peaks in the HMBC spectrum between H-1 of the glucose unit at δ 4.49 and C-3 (δC 91.47) of the aglycon and between H-2′′ of arabinose (δH 3.92) and C-1′′′ of the terminal rhamnose unit (δC 102.15) allowed the sequence of the trisaccharide chain at C-3 to be determined as shown in Figure 1. Thus 1 is 3β-O-R-L-rhamnopyranosyl-(1f2)-R-L-arabinopyranosyl-(1f2)-β-D-glucopyranosylurs-20(21)-en-28-oic acid 28-0-[β-D-2-O-sulfonylglucopyranosyl] ester. The NMR data indicated the same aglycon and sugar chain at C-3 for both compounds 1 and 2. The 1H-NMR spectrum of 2 was very similar to that of 1 except for the chemical shift of H-2′′′′ signal (δ 3.30 in 2, 4.20 in 1) of the glucose moiety at C-28. The carbon resonances of C-1′′′′ (+2.06 ppm), C-2′′′′ (-5.54 ppm), and C-3′′′′ (+0.77 ppm) of the glucose moiety at C-28 indicated the loss of the sulfate group at C-2′′′′. It is interesting to note that the loss of the -SO3H group at C-2′′′′ induced an upfield shift of the resonance of the C-16eq proton (-0.21 ppm) and a downfield shift of Me-27 (+0.03 ppm) on the aglycon of compound 2 with respect to compound 1. From the above data, 2 was determined to be 3β-OR-L-rhamnopyranosyl-(1f2)-R-L-arabinopyranosyl-(1f2)β-D-glucopyranosylurs-20(21)-en-28-oic acid 28-O-[βD-glucopyranosyl] ester. The negative FABMS spectrum of 3 showed a quasimolecular anion at m/z 749 [M - H]- and two main peaks due to the loss of a hexose and a deoxyhexose unit. The 1H-NMR spectrum in the aglycon part showed the presence of six singlet Me signals at δ 0.86, 0.90, 0.91, 0.95, 0.99, and 1.03, indicating a nor-oleane pentacyclic skeleton. An olefinic hydrogen resonating at comparatively lowfield (δ 5.68 br m) and the typical signal of an oxymethine at C-3 (δ 3.21, dd, J ) 12.0, 4.5 Hz) were

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also present. The 13C and DEPT 13C-NMR spectra showed 41 signals. The 29 13C-NMR resonances of the aglycon were recognized by subtracting the 12 sugar carbon resonances from the total spectrum. Some of them are peculiar for a 3-β-glycosylated olean-28-oic acid.20,21 The absence of the signal ascribable to Me-27 typically resonating at ca. δC 26.0 in the oleane skeleton, the olefinic resonances at δC 127.9 (CH) shifted to downfield by ca. 5 ppm and at δC 140.0 (C) shifted to upfield by ca. 3 ppm with respect to oleanolic acid,20,21 as well as the presence of a CH resonating at 55.5 (C14), indicated a 27-nor-olean-12-en-28-oic acid aglycon. The NMR spectra of 3 in the sugar region exhibited an ester-bonded anomeric signal at δH 5.42 (d, J ) 7.5 Hz) and δC 95.7 and a second ether-linked anomeric signal at δH 4.31 (d, J ) 7.9 Hz) and δC 106.6. Starting from these well-resolved anomeric proton signals the other proton sugar signals could be assigned by 1D TOCSY and 2D DQF-COSY spectra. In the 1D TOCSY spectrum of compound 3, H-1 signal at δ 5.42 showed connectivities to four methines and a methylene; the COSY spectrum established the proton sequence within this monosaccharide as H-1 (δ 5.42), H-2 (δ 3.28), H-3 (δ 3.43), H-4 (δ 3.57), H-5 (δ 3.37), H2-6 (δ 3.70 and 3.85). Similar observations on the other sugar residue and analysis of the 13C and DEPT 13C-NMR data (see Experimental Section) led to the identification of β-Dquinovopyranose linked at C-3 and β-D-glucopyranose linked at C-28 of the aglycon. Thus, 3 was the novel compound 3β-O-β-D-quinovopyranosyl-27-nor-olean-12en-28-oic acid 28-O-β-D-glucopyranosyl ester. The remaining saponins were identified by comparison of their NMR spectral data with those from the literature to be 3β-O-β-D-glucopyranosyl-quinovic acid 28-O-β-D-glucopyranosyl ester previously isolated from Guettarda platypoda,2 3β-O-[β-D-2-O-sulfonylglucopyranosyl]-quinovic acid 28-O-[β-D-glucopyranosyl) ester (zygophyloside G)3 and 3β-O-[β-D-2-O-sulfonylquinovopyranosyl]-quinovic acid 28-O-[β-D-glucopyranosyl) ester (zygophyloside E)4 previously isolated from Z. propinquum. Experimental Section General Experimental Procedures. A Bruker DRX-600 spectrometer operating at 599.19 MHz for 1H and 150.858 for 13C using the UXNMR software package was used for NMR measurements in CD3OD solutions. 2D experiments: 1H-1H DQF-COSY,11 inverse-detected 1H-13C HSQC12 and HMBC,13 and ROESY14 were obtained by employing the conventional pulse sequences as described previously.18,19,22 The selective excitation spectra, 1D TOCSY16 were acquired using waveform generator-based GAUSS shaped pulses, mixing times ranging from 100 to 120 ms, and a MLEV-17 spin-lock field of 10 kHz preceded by a 2.5-ms trim pulse. Optical rotations were measured on a PerkinElmer 141 polarimeter using a sodium lamp operating at 589 nm in 1% w/v solutions in MeOH. FABMS were recorded in a glycerol matrix in the negative ion mode on a VG ZAB instrument (XE atoms of energy of 2-6 KV). HPLC separations were performed with a Waters model 6000A pump equipped with a U6K injector and a model 401 refractive index detector. Plant Material. Z. gaetulum Emb. & Maire was collected in September 1992, near Zreouila 27 km from

134 Journal of Natural Products, 1998, Vol. 61, No. 1

Goulmine, southern Morocco. The plant was identified by Dr. A. Khaoudji of the Department de Biologie, Faculte´ des Sciences, Rabat, Morocco. A voucher specimen of the plant is deposited at the herbarium of Laboratoire de Chimie des Plantes et de Synthe`se Organique et Bioorganique, Faculte´ des Sciences, Rabat, Morocco. Extraction and Isolation. Air-dried and powdered aerial parts of Z. gaetulum (1 kg) were sequentially extracted at room temperature with petroleum ether, CHCl3, CHCl3-MeOH (9:1), MeOH, and H2O to afford 7.85, 7.03, 19.46, and 11.03 g of residue, respectively. Part of the MeOH residue (4 g) was chromatographed on a Sephadex LH-20 column (100 × 5 cm) eluting with MeOH. Fractions of 8 mL were collected and combined by TLC similarity [SiO2 plates, n-BuOH-AcOH-H2O (60:15:25) and CHCl3-MeOH-H2O (80:18:2)] into nine main fractions A-I. Fractions B (161 mg), E (520 mg), and I (329 mg) were further chromatographed by RPHPLC (µ-Bondapak C-18 column, 30 cm × 7.8 mm, flow rate 1.5 mL/min) with MeOH-H2O (54:46) as solvent system. Fraction B, containing the more polar glycosides, yielded compounds 1 (tR ) 4.5 min, 30.5 mg) and 2 (tR ) 9 min, 22 mg). Fractions E and I, containing the less polar glycosides, provided compounds 3 (tR ) 21 min, 15 mg), 3β-O-β-D-glucopyranosyl-quinovic acid 28-O-β-D-glucopyranosyl ester (tR ) 20.5 min, 15.5 mg), zygophyloside G (tR ) 15 min, 10 mg) and zygophyloside E (tR ) 18 min, 12 mg). The known saponins were identified by comparison of their NMR spectra with literature data.2-4 Compound 1: [R]25D +15.5; negative FABMS m/z [M - H]- 1137, [(M - H)- SO3]- 1057, [(M - H) - (SO3 + 162)]- 895; NMR data for the aglycon moiety are reported in Table 1; for the sugar moiety, in Table 3. Compound 2: [R]25D +18.1; negative FABMS m/z [M - H]- 1057, [(M - H)- 162]- 895; NMR data for the aglycon moiety are reported in Table 2; for the sugar moiety, in Table 3. Compound 3: [R]D25 +29.7; negative FABMS m/z [M - H]- 749, [(M - H) - 146]- 603, [(M - H) - 162]587, [(M - H) - (146 + 162)]- 441; 1H NMR δ 0.86 (3H, s, Me-23), 0.90 (3H, s, Me-26), 0.91 (3H, s, Me-30 or 29), 0.95 (3H, s, Me-29 or 30), 0.99 (3H, s, Me-25), 1.03 (3H, s, Me-24), 1.28 (3H, d, J ) 6.6 Hz, Qui-6), 3.21 (1H, dd, J ) 12.0, 4.5 Hz, H-3), 3.23 (1H, dd, J ) 7.9, 9.5 Hz, Qui-2), 3.28 (1H, dd, J ) 9.0, 7.6 Hz, Glc-2), 3.30 (1H, t, J ) 9.5 Hz, Qui-4), 3.33 (1H, dd, J ) 9.2, 6.5 Hz, Qui5), 3.37 (1H, ddd, J ) 9.0, 4.0, 2.5 Hz, Glc-5), 3.40 (1H, t, J ) 9.5 Hz, Qui-3), 3.43 (1H, t, J ) 9.0 Hz, Glc-3), 3.57 (1H, t, J ) 9.0 Hz, Glc-4), 3.70 (1H, dd, J ) 12.0, 4.0 Hz, Glc-6a), 3.85 (1H, dd, J ) 12.0, 2.5 Hz, Glc-6b), 4.31 (1H, d, J ) 7.9 Hz, Qui-1), 5.42 (1H, d, J ) 7.6 Hz,

Notes

Glc-1), 5.68 (IH, br m, H-12); 13C NMR δ 17.0 (Me-25), 18.1 (Qui-6), 18.4 (Me-26), 19.0 (Me-23), 19.2 (CH2-6), 24.1 (CH2-11), 24.38 (Me-30), 25.2 (CH2-16), 25.4 (CH215), 27.0 (CH2-2), 28.5 (Me-24), 30.1 (C-20), 31.0 (CH222), 33.6 (Me-29), 33.9 (CH2-7), 34.0 (CH2-21), 37.9 (C10), 39.8 (CH2-1), 40.1 (C-4), 40.6 (C-8), 43.0 (CH-18), 45.0 (CH2-19), 47.8 (C-17), 48.0 (CH-9), 55.5 (CH-14), 57.8 (CH-5), 62.5 (Glc-6), 71.2 (Qui-4), 73.1 (Glc-4), 74.0 (Glc-2), 75.9 (Qui-2), 77.1 (Qui-5), 78.0 (Glc-5), 78.4 (Qui3), 78.7 (Glc-3), 90.7 (CH-3), 95.7 (Glc-1), 106.6 (Qui-1), 127.9 (C-12), 140.0 (C-13), 176.0 (C-28). Solvolysis of 1. A solution of 1 in a 1:1 mixture of dioxane and pyridine (0.6 mL) was heated in a stoppered reaction vial at 120 °C for 4 h. The mixture was diluted with H2O and extracted with n-BuOH. The solvent was evaporated under reduced pressure and the residue analyzed by 1H NMR, which gave data identical with those of compound 2. References and Notes (1) Bellakhadar, J.; Claisse, R.; Fleurentin, J.; Younos, C. J. Ethnopharmacol. 1991, 35, 123-143. (2) Aquino, R.; De Simone, F.; Pizza, C.; Cerri, R.; De Mello, J. F. Phytochemistry 1988, 27, 2927-2930. (3) Ahmad, V. U.; Ghazala; Uddin, G. S.; Ali, M. S. Phytochemistry 1993, 33, 453-455. (4) Pollmann, K.; Gagel, S.; Elgamal, M. H. A.; Shaker, K. H.; Seifert, K. Phytochemistry 1997, 44, 485-489, and references cited therein. (5) Ahmad, V. U.; Ghazala; Uddin, G. S.; Bano, S. J. Nat. Prod. 1990, 53, 1193-1197. (6) Hassanean, H. A.; El-Hamouly, M. M. A.; El-Moghazy, S. A.; Bishay, D. W. Phytochemistry 1993, 33, 667-670, and references cited therein. (7) Khan, A. Q.; Ahmed, Z.; Kazmi, S. N. U. H.; Malik, A. J. Nat. Prod. 1988, 51, 925-928. (8) Siddiqui, S.; Begum, S.; Siddiqui, B. S.; Hafeez, F. J. Nat. Prod. 1989, 52, 57-62. (9) Yahara, S.; Ding, N.; Nohara, T.; Masuda, K.; Ageta, H. Phytochemistry 1997, 44, 131-135. (10) Ansari, A. A.; Kenne, L.; Rahaman, A. R. Phytochemistry 1987, 26, 1487-1490. (11) Bodenhausen, G.; Freeman, R.; Morris, G. A.; Neidermeyer, R.; Turner, J. J. Magn. Reson. 1977, 25, 559. (12) Bodenhausen, G.; Ruben, D. J. Chem. Phys. Lett. 1980, 69, 185186. (13) Martin, G. E.; Crouch, R. C. J. Nat. Prod. 1991, 54, 1-70. (14) Kessler, H.; Gresinger, C.; Kerssebaum, R.; Wagner, K.; Ernst, R. R. J. Am. Chem. Soc. 1987, 109, 607-609. (15) Misra, T. N.; Singh, R. S.; Pandey, H. S.; Prasad, C. J. Nat. Prod. 1995, 58, 1056-1058, and references cited therein. (16) Davis, D. G.; Bax, A. J. Am. Chem. Soc. 1985, 107, 7198-7199. (17) Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy, 3rd rev. ed.; VCH: Weinheim, 1989. (18) Piacente, S.; Pizza, C.; De Tommasi, N.; De Simone, F. J. Nat. Prod. 1995, 58, 512-519. (19) De Tommasi, N.; Piacente, S.; De Simone, F.; Pizza, C. J. Nat. Prod. 1993, 56, 1669-1675. (20) Doddrell, D. M.; Khong, P. W.; Lewis, K. G. Tetrahedron Lett. 1974, 2381-2385. (21) Seo, S.; Tomita, Y.; Tori, K. Tetrahedron Lett. 1975, 7-10. (22) De Tommasi, N.; De Simone, F.; Speranza, G.; Pizza, C. J. Agric. Food Chem. 1996, 44, 2020-2025.

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